Identification of diverse tumor endothelial cell populations in malignant glioma.

BACKGROUND: Glioblastoma is the most common and aggressive type of primary brain tumor, as most patients succumb to the disease less than two years after diagnosis. Critically, studies demonstrate that glioma recruits surrounding blood vessels, while some work suggests that tumor stem cells themselves directly differentiate into endothelial cells, yet the molecular and cellular dynamics of the endothelium in glioma are poorly characterized. The goal of this study was to establish molecular and morphological benchmarks for tumor associated vessels (TAVs) and tumor derived endothelial cells (TDECs) during glioblastoma progression. METHODS: Using In-Utero Electroporation and CRISPR/Cas9 genome engineering to generate a native, immunocompetent mouse model of glioma, we characterized vascular-tumor dynamics in three dimensions during tumor progression. We employed bulk and single-cell RNA-Sequencing to elucidate the relationship between TAVs and TDECs. We confirmed our findings in a patient derived orthotopic xenograft (PDOX) model. RESULTS: Using a mouse model of glioma, we identified progressive alteration of vessel function and morphogenesis over time. We also showed in our mouse model that TDECs are a rare subpopulation that contributes to vessels within the tumor, albeit to a limited degree. Furthermore, transcriptional profiling demonstrates that both TAVs and TDECs are molecularly distinct, and both populations feature extensive molecular heterogeneity. Finally, the distinct molecular signatures of these heterogeneous populations are also present in human glioma. CONCLUSIONS: Our findings show extensive endothelial heterogeneity within the tumor and tumor microenvironment and provide insights into the diverse cellular and molecular mechanisms that drive glioma vascularization and angiogenesis during tumorigenesis.

Inducible protein traps with dominant phenotypes for functional analysis of the Drosophila genome.

The Drosophila melanogaster genome has been extensively characterized, but there remains a pressing need to associate gene products with phenotypes, subcellular localizations, and interaction partners. A multifunctional, Minos transposon-based protein trapping system called Hostile takeover (Hto) was developed to facilitate in vivo analyses of endogenous genes, including live imaging, purification of protein complexes, and mutagenesis. The Hto transposon features a UAS enhancer with a basal promoter, followed by an artificial exon 1 and a standard 5' splice site. Upon GAL4 induction, exon 1 can splice to the next exon downstream in the flanking genomic DNA, belonging to a random target gene. Exon 1 encodes a dual tag (FLAG epitope and mCherry red fluorescent protein), which becomes fused to the target protein. Hto was mobilized throughout the genome and then activated by eye-specific GAL4; an F1 screen for abnormal eye phenotypes was used to identify inserts that express disruptive fusion proteins. Approximately 1.7% of new inserts cause eye phenotypes. Of the first 23 verified target genes, 21 can be described as regulators of cell biology and development. Most are transcription factor genes, including AP-2, CG17181, cut, klu, mamo, Sox102F, and sv. Other target genes [l(1)G0232, nuf, pum, and Syt4] make cytoplasmic proteins, and these lines produce diverse fluorescence localization patterns. Hto permits the expression of stable carboxy-terminal subfragments of proteins, which are rarely tested in conventional genetic screens. Some of these may disrupt specific cell pathways, as exemplified by truncated forms of Mastermind and Nuf.

Targeted disruption of Hotair leads to homeotic transformation and gene derepression.

Long noncoding RNAs (lncRNAs) are thought to be prevalent regulators of gene expression, but the consequences of lncRNA inactivation in vivo are mostly unknown. Here, we show that targeted deletion of mouse Hotair lncRNA leads to derepression of hundreds of genes, resulting in homeotic transformation of the spine and malformation of metacarpal-carpal bones. RNA sequencing and conditional inactivation reveal an ongoing requirement of Hotair to repress HoxD genes and several imprinted loci such as Dlk1-Meg3 and Igf2-H19 without affecting imprinting choice. Hotair binds to both Polycomb repressive complex 2, which methylates histone H3 at lysine 27 (H3K27), and Lsd1 complex, which demethylates histone H3 at lysine 4 (H3K4) in vivo. Hotair inactivation causes H3K4me3 gain and, to a lesser extent, H3K27me3 loss at target genes. These results reveal the function and mechanisms of Hotair lncRNA in enforcing a silent chromatin state at Hox and additional genes.